The long-term goal of this research is to gain scientific knowledge that can be used to improve outcome in patients with head and neck squamous cell carcinoma (HNSCC). HNSCC is one of the 10 most common cancers worldwide, and the incidence of HNSCC located in the oropharyngeal sites is increasing. This trend has been attributed to HNSCC associated with papillomavirus (HPV), which has been recognized to be a distinct biological entity from the HPV-negative counterpart that is primarily smoking associated/chemically induced HNSCC. It is well documented that HPV-positive oropharyngeal cancer patients experience significantly lower locoregional recurrence and significantly higher overall survival in comparison with HPV-negative patients, especially among those who received radiation therapy. In this R21 application, we hypothesize that the survival advantage in patients with HPV-positive HNSCC results from increased radiosensitivity mediated through differential regulation of tumor metabolic pathways between HPV-associated and smoking-associated cancers. This hypothesis is based on studies from others as well as our preliminary data. A previous study showed that transfection of the dominant-negative p53 mutant into oral keratinocytes, mimicking smoking-associated cancer, results in upregulation of hypoxia signaling (HIF1) pathway, which in turn could increase glucose metabolism, whereas introduction of HPV16-E6 into those cells leads to downregulation of HIF-1. Importantly, recent studies suggested an association between HIF1-medicated glucose metabolism and radioresistance. Consistently, our preliminary data showed that HPV-negative HNSCC cells express higher levels of hexokinase, the rate-limiting enzyme in glycolysis, whereas HPV-positive HNSCC cells express low levels of hexokinase and high levels of cytochrome c oxidase, a key enzyme in the mitochondrial respiratory pathway. Taken together, our working hypothesis is that HPV-positive HNSCC cells rely on mitochondrial respiration with decreased glucose metabolism, whereas smoking-associated/chemically-induced HNSCC cells heavily rely on glycolytic pathways. To address this hypothesis and to develop a biomarker-based radiation sensitizer, we propose the following specific aims: (1) To investigate the relationship between the HPV status and tumor metabolism in selected oropharyngeal cancer cell lines and to elucidate the role of HPV16 oncoproteins in tumor metabolism in vitro; (2) To evaluate the effect of pharmacological manipulation of metabolic pathways on radiation sensitivity of HNSCC cells in vitro and in vivo animal model; (3) To examine expression levels tumor metabolic pathway genes in clinical oropharyngeal cancer samples in relation to HPV status and overall survival; and (4) To explore if overall survival is worse in diabetic HNSCC patients than in non-diabetic HNSCC patients, especially in relation to radiation therapy. Completion of this exploratory R21 application will help us understand molecular basis for HPV-associated radiosensitivity and provide important information for further patient stratification and the design of biomarker-driven therapy.